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Tina Dalianis - One of the best experts on this subject based on the ideXlab platform.
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2016Co-Authors: Karin Tegerstedt, Shirin Heidari, Andrea Vlastos, See Profile, Tina DalianisAbstract:Murine Polyomavirus virus-like particles (VLPs) as vectors for gene and immune therapy and vaccines against viral infections and cance
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Immunotherapy for Polyomaviruses: opportunities and challenges.
Immunotherapy, 2012Co-Authors: Tina DalianisAbstract:Polyomaviruses are small DNA viruses present in mammals and birds, and in 1953 the first one to be described was Murine Polyomavirus. It was not until 1971 that the first two human Polyomaviruses (HPyVs), BK virus and JC virus, were discovered and found to be common in humans, but only associated with disease in severely immunosuppressed patients. Since 2007, seven new HPyVs have been identified: KI Polyomavirus, WU Polyomavirus, Merkel cell Polyomavirus, HPyV6, HPyV7, trichodyplasia spinulosa Polyomavirus and HPyV9. Notably, Merkel cell Polyomavirus was detected in Merkel cell cancer, a tumor mainly found in elderly and immunocompromised individuals, while trichodyplasia spinulosa Polyomavirus was found in trichodyplasia spinulosa, a skin disorder observed only in immunosuppressed individuals. Consequently, many Polyomaviruses cause problems in immunosuppressed individuals. This review deals with these issues, and the potential of the capsid protein VP1 to form virus-like particles for use as vaccines against Polyomavirus infections.
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Murine Polyomavirus virus-like particles carrying full-length human PSA protect BALB/c mice from outgrowth of a PSA expressing tumor.
PloS one, 2011Co-Authors: Mathilda Eriksson, Karin Tegerstedt, Tina Dalianis, Kalle Andreasson, Joachim Weidmann, Kajsa Lundberg, Torbjörn RamqvistAbstract:Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed Murine Polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto Murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4+ and CD8+ cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4+ and CD8+ cells with a low induction of anti-VLP antibodies.
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Murine Polyomavirus virus like particles carrying full length human psa protect balb c mice from outgrowth of a psa expressing tumor
PLOS ONE, 2011Co-Authors: Mathilda Eriksson, Karin Tegerstedt, Tina Dalianis, Kalle Andreasson, Joachim Weidmann, Kajsa Lundberg, Torbjörn RamqvistAbstract:Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed Murine Polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto Murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4+ and CD8+ cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4+ and CD8+ cells with a low induction of anti-VLP antibodies.
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Lessons from Immune Responses and Vaccines against Murine Polyomavirus Infection and Polyomavirus-induced Tumours Potentially Useful for Studies on Human Polyomaviruses
Anticancer research, 2010Co-Authors: Torbjörn Ramqvist, Tina DalianisAbstract:During 2007-2008, three new human Polyomaviruses, KI, WU and Merkel cell cancer Polyomaviruses have been discovered, of which the latter has also been identified in a human tumour. This development revives the interest in both human and animal Polyomaviruses and their potential role in tumour development and disease particularly in immune suppressed individuals. Murine Polyomavirus (MPyV) has in the past been used for acquiring knowledge of transformation mechanisms in vitro, as well as in immunological studies with regard to virus-induced tumour development in the natural host of the virus. Here we summarize some of the accumulated knowledge achieved in the MPyV field in view of the balance between tumour virus, the immune system and tumour development, and discuss this in relation to infections with human Polyomaviruses. We also present how virus-like particles (VLPs) and gluthatione-S-transferase VP1 can be used for vaccination against the same tumour virus not only in mice with a well-functioning immune system, but also in immune suppressed mice.
Thilo Stehle - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and
2016Co-Authors: Michael H. C. Buch, Robert L. Garcea, Manuel A. Liaci, Samantha D. O’hara, Ursula Neu, Thilo StehleAbstract:Murine Polyomavirus (MuPyV) causes tumors of various origins in newborn mice and ham-sters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell sur-face. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathoge-nicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglio-side deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosyn
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Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity
PLoS pathogens, 2015Co-Authors: Michael H. C. Buch, Robert L. Garcea, Samantha D. O’hara, Ursula Neu, A. Manuel Liaci, Thilo StehleAbstract:Murine Polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.
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Glycan receptors of the Polyomaviridae: structure, function, and pathogenesis.
Current opinion in virology, 2014Co-Authors: Samantha D. O’hara, Thilo Stehle, Robert L. GarceaAbstract:Multiple glycans have been identified as potential cell surface binding motifs for Polyomaviruses (PyVs) using both crystallographic structural determinations and in vitro binding assays. However, binding alone does not necessarily imply that a glycan is a functional receptor, and confirmation that specific glycans are important for infection has proved challenging. In vivo analysis of Murine Polyomavirus (MPyV) infection has shown that subtle alterations in PyV–glycan interactions alone can result in dramatic changes in pathogenicity, implying that similar effects will be found for other PyVs. Our discussion will review the assays used for determining virus–glycan binding, and how these relate to known PyV tropism and pathogenesis.
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Structures of the Major Capsid Proteins of the Human Karolinska Institutet and Washington University Polyomaviruses
Journal of virology, 2011Co-Authors: Ursula Neu, Robert L. Garcea, Jianbo Wang, Dennis Macejak, Thilo StehleAbstract:The Karolinska Institutet and Washington University Polyomaviruses (KIPyV and WUPyV, respectively) are recently discovered human viruses that infect the respiratory tract. Although they have not yet been linked to disease, they are prevalent in populations worldwide, with initial infection occurring in early childhood. Polyomavirus capsids consist of 72 pentamers of the major capsid protein viral protein 1 (VP1), which determines antigenicity and receptor specificity. The WUPyV and KIPyV VP1 proteins are distant in evolution from VP1 proteins of known structure such as simian virus 40 or Murine Polyomavirus. We present here the crystal structures of unassembled recombinant WUPyV and KIPyV VP1 pentamers at resolutions of 2.9 and 2.55 A, respectively. The WUPyV and KIPyV VP1 core structures fold into the same β-sandwich that is a hallmark of all Polyomavirus VP1 proteins crystallized to date. However, differences in sequence translate into profoundly different surface loop structures in KIPyV and WUPyV VP1 proteins. Such loop structures have not been observed for other Polyomaviruses, and they provide initial clues about the possible interactions of these viruses with cell surface receptors.
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structural basis of gm1 ganglioside recognition by simian virus 40
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Ursula Neu, Thilo Stehle, Karin Woellner, Guenter GauglitzAbstract:Simian virus 40 (SV40) has been a paradigm for understanding attachment and entry of nonenveloped viruses, viral DNA replication, and virus assembly, as well as for endocytosis pathways associated with caveolin and cholesterol. We find by glycan array screening that SV40 recognizes its ganglioside receptor GM1 with a quite narrow specificity, but isothermal titration calorimetry shows that individual binding sites have a relatively low affinity, with a millimolar dissociation constant. The high-resolution crystal structure of recombinantly produced SV40 capsid protein, VP1, in complex with the carbohydrate portion of GM1, reveals that the receptor is bound in a shallow solvent-exposed groove at the outer surface of the capsid. Through a complex network of interactions, VP1 recognizes a conformation of GM1 that is the dominant one in solution. Analysis of contacts provides a structural basis for the observed specificity and suggests binding mechanisms for additional physiologically relevant GM1 variants. Comparison with Murine Polyomavirus (Polyoma) receptor complexes reveals that SV40 uses a different mechanism of sialic acid binding, which has implications for receptor binding of human Polyomaviruses. The SV40–GM1 complex reveals a parallel to cholera toxin, which uses a similar cell entry pathway and binds GM1 in the same conformation.
ANTON PETER JACOB MIDDELBERG - One of the best experts on this subject based on the ideXlab platform.
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Chimeric Murine Polyomavirus virus-like particles induce Plasmodium antigen-specific CD8+ T cell and antibody responses
Frontiers in cellular and infection microbiology, 2019Co-Authors: David J. Pattinson, Yap P Chuan, ANTON PETER JACOB MIDDELBERG, Simon H. Apte, Nani Wibowo, Tania Rivera-hernandez, Penny Groves, Linda H.l. Lua, Denise L. DoolanAbstract:An effective vaccine against the Plasmodium parasite is likely to require the induction of robust antibody and T cell responses. Chimeric virus-like particles are an effective vaccine platform for induction of antibody responses, but their capacity to induce robust cellular responses and cell-mediated protection against pathogen challenge has not been established. To evaluate this, we produced chimeric constructs using the Murine Polyomavirus structural protein with surface-exposed CD8+ or CD4+ T cell or B cell repeat epitopes derived from the Plasmodium yoelii circumsporozoite protein, and assessed immunogenicity and protective capacity in a Murine model. Robust CD8+ T cell responses were induced by immunization with the chimeric CD8+ T cell epitope virus-like particles, however CD4+ T cell responses were very low. The B cell chimeric construct induced robust antibody responses but there was no apparent synergy when T cell and B cell constructs were administered as a pool. A heterologous prime/boost regimen using plasmid DNA priming followed by a VLP boost was more effective than homologous VLP immunization for cellular immunity and protection. These data show that chimeric Murine Polyomavirus virus-like particles are a good platform for induction of CD8+ T cell responses as well as antibody responses.
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Integrated molecular and bioprocess engineering for bacterially‐produced immunogenic modular virus‐like particle vaccine displaying 18 kDa rotavirus antigen
Biotechnology and Bioengineering, 2016Co-Authors: Alemu Tekewe, ANTON PETER JACOB MIDDELBERGAbstract:A high global burden of rotavirus disease and the unresolved challenges with the marketed rotavirus vaccines, particularly in the developing world, have ignited efforts to develop virus-like particle (VLP) vaccines for rotavirus. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising alternatives in reducing process complexity and cost. In this study, integrated molecular and bioprocess engineering approaches were used to simplify the production of modular Murine Polyomavirus capsomeres and VLPs presenting a rotavirus 18kDa VP8* antigen. A single construct was generated for dual expression of non-tagged Murine Polyomavirus capsid protein VP1 and modular VP1 inserted with VP8*, for co-expression in Escherichia coli. Co-expressed proteins assembled into pentameric capsomeres in E. coli. A selective salting-out precipitation and a polishing size exclusion chromatography step allowed the recovery of stable modular capsomeres from cell lysates at high purity, and modular capsomeres were successfully translated into modular VLPs when assembled in vitro. Immunogenicity study in mice showed that modular capsomeres and VLPs induced high levels of VP8*-specific antibodies. Our results demonstrate that a multipronged synthetic biology approach combining molecular and bioprocess engineering enabled simple and low-cost production of highly immunogenic modular capsomeres and VLPs presenting conformational VP8* antigenic modules. This strategy potentially provides a cost-effective production route for modular capsomere and VLP vaccines against rotavirus, highly suitable to manufacturing economics for the developing world. Biotechnol. Bioeng. 2017;114: 397-406. (c) 2016 Wiley Periodicals, Inc.
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Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus‐like particle
Protein Science, 2016Co-Authors: Alemu Tekewe, Natalie K. Connors, ANTON PETER JACOB MIDDELBERGAbstract:Virus-like particles (VLPs) and capsomere subunits have shown promising potential as safe and effective vaccine candidates. They can serve as platforms for the display of foreign epitopes on their surfaces in a modular architecture. Depending on the physicochemical properties of the antigenic modules, modularization may affect the expression, solubility and stability of capsomeres, and VLP assembly. In this study, three module designs of a rotavirus hydrophobic peptide (RV10) were synthesized using synthetic biology. Among the three synthetic modules, modularization of the Murine Polyomavirus VP1 with a single copy of RV10 flanked by long linkers and charged residues resulted in the expression of stable modular capsomeres. Further employing the approach of module titration of RV10 modules on each capsomere via Escherichia coli co-expression of unmodified VP1 and modular VP1-RV10 successfully translated purified modular capomeres into modular VLPs when assembled in vitro. Our results demonstrate that tailoring the physicochemical properties of modules to enhance modular capsomeres stability is achievable through synthetic biology designs. Combined with module titration strategy to avoid steric hindrance to intercapsomere interactions, this allows bioprocessing of bacterially produced in vitro assembled modular VLPs.
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molecular energetics in the capsomere of virus like particle revealed by molecular dynamics simulations
Journal of Physical Chemistry B, 2013Co-Authors: Lin Zhang, Natalie K. Connors, Yap P Chuan, Ronghong Tang, ANTON PETER JACOB MIDDELBERGAbstract:Virus-like particles (VLPs) are highly organized nanoparticles that have great potential in vaccinology, gene therapy, drug delivery, and materials science. However, the application of VLPs is hindered by obstacles in their design and production due to low efficiency of self-assembly. In the present study, all-atom (AA) molecular dynamics (MD) simulations coupled with the molecular mechanics-Poisson–Boltzmann surface area (MM-PBSA) method are utilized to examine the molecular interactions in the capsomere of a Murine Polyomavirus (MPV) VLP. It is found that both low ionic strength and the intracapsomere disulfide bonds are favorable for maintaining a stable capsomere. Simulation results examining the effects of solution conditions on the stabilization of a capsomere were verified by calorimetry experiments. Simulation results of free energy decomposition indicate that hydrophobic interaction is favorable for the formation of a capsomere, whereas electrostatic interaction is unfavorable. With increasing io...
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Quantitative characterization of virus‐like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy
Biotechnology and bioengineering, 2009Co-Authors: Leonard F. Pease, Daniel I Lipin, Linda H.l. Lua, De-hao Tsai, Michael R. Zachariah, Michael J. Tarlov, ANTON PETER JACOB MIDDELBERGAbstract:Here we characterize virus-like particles (VLPs) by three very distinct, orthogonal, and quantitative techniques: electrospray differential mobility analysis (ES-DMA), asymmetric flow field-flow fractionation with multi-angle light scattering detection (AFFFF-MALS) and transmission electron microscopy (TEM). VLPs are biomolecular particles assembled from viral proteins with applications ranging from synthetic vaccines to vectors for delivery of gene and drug therapies. VLPs may have polydispersed, multimodal size distributions, where the size distribution can be altered by subtle changes in the production process. These three techniques detect subtle size differences in VLPs derived from the non-enveloped Murine Polyomavirus (MPV) following:(i) functionalization of the surface of VLPs with an influenza viral peptide fragment; (ii) packaging of foreign protein internally within the VLPs; and (iii) packaging of genomic DNA internally within the VLPs. These results demonstrate that ES-DMA and AFFFF-MALS are able to quantitatively determine VLP size distributions with greater rapidity and statistical significance than TEM, providing useful technologies for product development and process analytics.
Torbjörn Ramqvist - One of the best experts on this subject based on the ideXlab platform.
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Murine Polyomavirus virus like particles carrying full length human psa protect balb c mice from outgrowth of a psa expressing tumor
PLOS ONE, 2011Co-Authors: Mathilda Eriksson, Karin Tegerstedt, Tina Dalianis, Kalle Andreasson, Joachim Weidmann, Kajsa Lundberg, Torbjörn RamqvistAbstract:Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed Murine Polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto Murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4+ and CD8+ cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4+ and CD8+ cells with a low induction of anti-VLP antibodies.
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Murine Polyomavirus virus-like particles carrying full-length human PSA protect BALB/c mice from outgrowth of a PSA expressing tumor.
PloS one, 2011Co-Authors: Mathilda Eriksson, Karin Tegerstedt, Tina Dalianis, Kalle Andreasson, Joachim Weidmann, Kajsa Lundberg, Torbjörn RamqvistAbstract:Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed Murine Polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto Murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4+ and CD8+ cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4+ and CD8+ cells with a low induction of anti-VLP antibodies.
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Lessons from Immune Responses and Vaccines against Murine Polyomavirus Infection and Polyomavirus-induced Tumours Potentially Useful for Studies on Human Polyomaviruses
Anticancer research, 2010Co-Authors: Torbjörn Ramqvist, Tina DalianisAbstract:During 2007-2008, three new human Polyomaviruses, KI, WU and Merkel cell cancer Polyomaviruses have been discovered, of which the latter has also been identified in a human tumour. This development revives the interest in both human and animal Polyomaviruses and their potential role in tumour development and disease particularly in immune suppressed individuals. Murine Polyomavirus (MPyV) has in the past been used for acquiring knowledge of transformation mechanisms in vitro, as well as in immunological studies with regard to virus-induced tumour development in the natural host of the virus. Here we summarize some of the accumulated knowledge achieved in the MPyV field in view of the balance between tumour virus, the immune system and tumour development, and discuss this in relation to infections with human Polyomaviruses. We also present how virus-like particles (VLPs) and gluthatione-S-transferase VP1 can be used for vaccination against the same tumour virus not only in mice with a well-functioning immune system, but also in immune suppressed mice.
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Murine Polyomavirus tumour specific transplantation antigens and viral persistence in relation to the immune response, and tumour development.
Seminars in cancer biology, 2009Co-Authors: Torbjörn Ramqvist, Tina DalianisAbstract:Murine Polyomavirus (MPyV) has been instrumental in the studies of transformation in vitro as well as for in vivo studies of virus induced tumour development, and for the studies of immune responses against a tumour virus in its natural host. Moreover, it has been extremely useful for unravelling different aspects of DNA replication, transcription, transformation, cell cycle regulation, and assembly of virus-like particles (VLPs). Here, we review how MPyV has also supplied us with information regarding: the molecular nature of a tumour specific transplantation antigen, knowledge on viral persistence and tumour development in its natural host, as well as experience regarding vaccination against infection with a tumour virus.
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Murine Polyomavirus virus-like particles as vectors for gene and immune therapy and as vaccines
Future Virology, 2007Co-Authors: Torbjörn Ramqvist, Kalle Andreasson, Tina DalianisAbstract:Polyomavirus virus-like particles (VLPs) can be produced free from viral genes and used as vectors for gene and immune therapy and as vaccines. For large-scale VLP manufacture, the major viral capsid protein (VP)1, is produced in a baculovirus insect cell system, Escherichia coli or yeast, and will self-assemble into VLPs under appropriate conditions. Murine Polyomavirus (MPyV) VLP vaccination prevents primary MPyV infection and outgrowth of some MPyV-tumors in mice. Furthermore, MPyV-VLPs bind and introduce eukaryotic DNA into various cells in vitro and in vivo, while MPyV-VLPs containing fusion proteins between capsid proteins VP1, -2 or -3 and selected antigens can be used as vaccines. Similar findings apply to other Polyomavirus VLPs. In summary, Polyomavirus VLPs are useful vectors for immune and gene therapy and as vaccines, and different Polyomavirus VLPs can be used for prime-boost therapy.
Brian Schaffhausen - One of the best experts on this subject based on the ideXlab platform.
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A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling.
Journal of virology, 2017Co-Authors: Deborah Denis, Cecile Rouleau, Brian SchaffhausenAbstract:Middle T antigen (MT), the principal oncoprotein of Murine Polyomavirus, transforms by association with cellular proteins. Protein phosphatase 2A (PP2A), YAP, Src family tyrosine kinases, Shc, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ1) have all been implicated in MT transformation. Mutant dl1015, with deletion of residues 338 to 347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets Src homology domain 3 (SH3) domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation defective. Extensive analysis of the E349K mutant is described here. Similar to wild-type MT, the E349K mutant associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase, and PLCγ1. The E349K mutant was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by coimmunoprecipitation, showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of the E349K mutant with PI3K, cells expressing the mutant failed to elevate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in mutant-expressing cells. These results indicate a novel unsuspected aspect to PI3K control. IMPORTANCE The gene coding for middle T antigen (MT) is the Murine Polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like the wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like the wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect.
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Papillomavirus E7 Oncoproteins Share Functions with Polyomavirus Small T Antigens
Journal of virology, 2014Co-Authors: Elizabeth A. White, Justin H. Hwang, Thomas M. Roberts, Brian Schaffhausen, Rebecca E. Kramer, Arun T. Pores Fernando, Nana Naetar, William C. Hahn, David M. Livingston, Peter M. HowleyAbstract:Many of the small DNA tumor viruses encode transforming proteins that function by targeting critical cellular pathways involved in cell proliferation and survival. In this study, we have examined whether some of the functions of the Polyomavirus small T antigens (ST) are shared by the E6 and E7 oncoproteins of two oncogenic papillomaviruses. Using three different assays, we have found that E7 can provide some simian virus 40 (SV40) or Murine Polyomavirus (PyV) ST functions. Both human papillomavirus 16 (HPV16) and bovine papillomavirus (BPV1) E7 proteins are capable of partially substituting for SV40 ST in a transformation assay that also includes SV40 large T antigen, the catalytic subunit of cellular telomerase, and oncogenic Ras. Like SV40 ST, HPV16 E7 has the ability to override a quiescence block induced by mitogen deprivation. Like PyV ST, it also has the ability to inhibit myoblast differentiation. At least two of these activities are dependent upon the interaction of HPV16 E7 with retinoblastoma protein family members. For small T antigens, interaction with PP2A is needed for each of these functions. Even though there is no strong evidence that E6 or E7 share the ability of small T to interact with PP2A, E7 provides these functions related to cellular transformation. IMPORTANCE DNA tumor viruses have provided major insights into how cancers develop. Some viruses, like the human papillomaviruses, can cause cancer directly. Both the papillomaviruses and the Polyomaviruses have served as tools for understanding pathways that are often perturbed in cancer. Here, we have compared the functions of transforming proteins from several DNA tumor viruses, including two papillomaviruses and two Polyomaviruses. We tested the papillomavirus E6 and E7 oncoproteins in three functional assays and found that E7 can provide some or all of the functions of the SV40 small T antigen, another well-characterized oncoprotein, in two of these assays. In a third assay, papillomavirus E7 has the same effect as the Murine Polyomavirus small T protein. In summary, we report several new functions for the papillomavirus E7 proteins, which will contribute new insights into the roles of viruses in cancer and the cellular pathways they perturb in carcinogenesis.
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Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.
PLoS pathogens, 2013Co-Authors: Pubali Banerjee, Rowena Dejesus, Ole Gjoerup, Brian SchaffhausenAbstract:Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with Murine Polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.
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Polyomavirus large T antigen binds symmetrical repeats at the viral origin in an asymmetrical manner.
Journal of virology, 2013Co-Authors: Celia J. Harrison, Brian Schaffhausen, Tao Jiang, Pubali Banerjee, Gretchen Meinke, Claudia M. D’abramo, Andrew BohmAbstract:Polyomaviruses have repeating sequences at their origins of replication that bind the origin-binding domain of virus-encoded large T antigen. In Murine Polyomavirus, the central region of the origin contains four copies (P1 to P4) of the sequence G(A/G)GGC. They are arranged as a pair of inverted repeats with a 2-bp overlap between the repeats at the center. In contrast to simian virus 40 (SV40), where the repeats are nonoverlapping and all four repeats can be simultaneously occupied, the crystal structure of the four central Murine Polyomavirus sequence repeats in complex with the Polyomavirus origin-binding domain reveals that only three of the four repeats (P1, P2, and P4) are occupied. Isothermal titration calorimetry confirms that the stoichiometry is the same in solution as in the crystal structure. Consistent with these results, mutation of the third repeat has little effect on DNA replication in vivo. Thus, the apparent 2-fold symmetry within the DNA repeats is not carried over to the protein-DNA complex. Flanking sequences, such as the AT-rich region, are known to be important for DNA replication. When the orientation of the central region was reversed with respect to these flanking regions, the origin was still able to replicate and the P3 sequence (now located at the P2 position with respect to the flanking regions) was again dispensable. This highlights the critical importance of the precise sequence of the region containing the pentamers in replication.
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Comparisons between Murine Polyomavirus and Simian Virus 40 Show Significant Differences in Small T Antigen Function
Journal of virology, 2011Co-Authors: Shaida Andrabi, Justin H. Hwang, Jennifer Kean Choe, Thomas M. Roberts, Brian SchaffhausenAbstract:Although members of a virus family produce similar gene products, those products may have quite different functions. Simian virus 40 (SV40) large T antigen (LT), for example, targets p53 directly, but Murine Polyomavirus LT does not. SV40 small T antigen (SVST) has received considerable attention because of its ability to contribute to transformation of human cells. Here, we show that there are major differences between SVST and Polyomavirus small T antigen (POLST) in their effects on differentiation, transformation, and cell survival. Both SVST and POLST induce cell cycle progression. However, POLST also inhibits differentiation of 3T3-L1 preadipocytes and C2C12 myoblasts. Additionally, POLST induces apoptosis of mouse embryo fibroblasts. SVST reduces the proapoptotic transcriptional activity of FOXO1 through phosphorylation. On the other hand, SVST complements large T antigen and Ras for the transformation of human mammary epithelial cells (HMECs), but POLST does not. Mechanistically, the differences between SVST and POLST may lie in utilization of protein phosphatase 2A (PP2A). POLST binds both Aα and Aβ scaffolding subunits of PP2A while SVST binds only Aα. Knockdown of Aβ could mimic POLST-induced apoptosis. The two small T antigens can target different proteins for dephosphorylation. POLST binds and dephosphorylates substrates, such as lipins, that SVST does not.
